The long-term goal of this proposal is to elucidate molecular mechanism(s) by which Wingless (Wg) morphogen gradient and signaling is regulated during development. Drosophila Wg encodes an evolutionary conserved glycoprotein of the Wnt family. Aberrant Wg/Wnt signaling activity underlies a number of human developmental disorders and contributes to a variety of cancers. Thus, elucidation of Wg/Wnt signaling pathway will provide new insights into the mechanisms of related human diseases. Wg acts as a critical regulator in many developmental processes. In a variety of developmental contexts, Wg can function as a short-range organizer and long-range morphogen that acts several cell diameters away to pattern a field of tissue in a concentration dependent manner. Although the components of Wg/Wnt signaling pathway in its receiving cells have been well characterized over the past 15 years, it remains poorly understood how the mature form of Wg is synthesized, secreted and travels to its receiving cells to form concentration gradient. The main focus of this application is to define the molecular mechanisms controlling Wg protein secretion, movement and its subsequent distributions. This is an application for continuing support. In the previous grant period, we have taken genetic and cell biology approaches in Drosophila to address the roles of heparan sulfate proteoglycans (HSPG) in Wg gradient formation and its signaling activity. Our results have established the essential roles of Drosophila HSPGs Dally and Dally-like (Dip) in Wg morphogen gradient formation. The HSPGs Dally and Dip controls Wg morphogen movement by a restrict diffusion mechanism. In the next grant period, we will focus our studies to address four related issues. First, we will determine the dual roles of Pore in Wg secretion and palmitoylation (Aim1). Second, we will examine the molecular mechanisms of retromer in Wg secretion and distribution (Aim 2). Third, we will determine the mechanisms by which palmitoylation controls Wg signaling and distribution (Aim3). Finally, we will dissect the mechanisms by which Notum modulates the Wg signaling and distribution (Aim4). Altogether, our genetic and biochemical analyses of HSPGs will elucidate the role of HSPGs in regulating morphogen gradient formation and signaling of Wg proteins that play major roles in both developmental processes and oncogenesis. Since Wg signaling transduction pathway is conserved between Drosophila and human, the outcomes of this project will clearly provide new insights into the mechanisms of the disease processes including cancers, which are associated with Wg signaling and HSPGs.